July 1, 2010 (Vol. 30, No. 13)

Patricia F. Fitzpatrick Dimond Ph.D. Technical Editor of Clinical OMICs President of BioInsight Communications

Generating Functional Tissue Constructs that Avoid Immunogenicity Remains Elusive but Not for Long

Biological scaffolding, either alone or in combination with cells for tissue regeneration, has the potential to revolutionize the repair of injured tissues and to replace tissues lost through disease and injury. Requirements for scaffolding include biocompatibility, hemocompatability, and the use of nontoxic materials that are durable, functional, and able to support cell growth. In addition, repair or replacement cells or tissues should not provoke immune reactions in the host.

Bioscaffolding materials may originate from multiple animal, tissue, and cell sources, as well as from synthetic polymers.  While many types of matrices have been successfully applied in the clinic, fundamental questions remain about how to combine and manipulate cells with scaffolding to generate functional tissue constructs that avoid immunogenicity.

At McGowan Institute’s “Symposium on Biologic Scaffolds for Regenerative Medicine” held recently in Boulder, CO, academic and industry scientists discussed topics ranging from peptide-based scaffolds for nerve tissue to tissue engineering via self assembly.

Approaches to scaffolding technology that were discussed at the meeting ranged in complexity from tissue repair products consisting of bovine type 1 collagen knee implants to the highly complex such as the use of whole acellular lung matrix (ACM) to support the development of engineered lung tissue from embryonic stem cells.

John Dichiara, svp of clinical and regulatory affairs at ReGen Biologics, discussed the clinical performance of ReGen’s collagen surgical mesh scaffold used for the reinforcement and repair of medial meniscus injuries. ReGen’s Menaflex device, cleared for marketing in the U.S. in 2008, was approved in the EU for use in repair of the medial meniscus in 2000 and the lateral meniscus in 2006. To date, more than 3,000 devices have reportedly been distributed.

Menaflex consists of chemically crosslinked, porous type 1 collagen sourced from bovine achilles tendons. The device is anatomically shaped like a human meniscus and is intended, when sutured to the meniscus rim, to act as a tissue scaffold and fill the void left after meniscus loss. The device provides a scaffold for meniscus-like fibrochondrocytic matrix production and the newly formed tissue integrates into the host meniscal rim.

Recent study results showed statistically significant improvement in pain, function, self-assessment, and activity levels from baseline at a mean of five years with Menaflex as opposed to partial meniscectomy. Although these results were not statistically superior to partial meniscectomy which is one of the most successful orthopedic surgical procedures, Menaflex patients, DiChiara said, had the additional benefit of approximately 70% more tissue within the meniscus to potentially protect the articular surfaces from degradation.

Tissue Replacements

Tissue engineers working at the laboratory for stem cells and tissue engineering at Columbia University’s Fu Foundation School of Engineering are applying a biomimetic approach to developing tissue replacements. Donald O. Freytes, Ph.D., a postdoctoral fellow working in the laboratory of Gordana Vunjak-Novakovic, Ph.D., professor of biomedical engineering at Columbia, described the use of decellularized matrices for myocardium and bone replacement.

Dr. Freytes explained that bone reconstructions, such as craniofacial reconstruction, often involve “autologous tissue grafting, a method limited by harvesting difficulties, donor site morbidity, and the clinicians’ ability to contour delicate 3-D shapes.” The availability of personalized bone grafts engineered from the patient’s own stem cells, he said “would revolutionize the way we currently treat defects.”

For their bone reconstruction model, the scientists generated anatomically shaped scaffolds in the exact shape of the human temperomandibular joint (TMJ) bone from decellularized trabecular bone using digitized clinical images, seeded with human mesenchymal stem cells, and cultured with interstitial flow of culture medium.

A bioreactor with an anatomical culture chamber was designed for controllable perfusion throughout the engineered construct in collaboration with Warren L. Grayson, Ph.D., assistant professor of biomedical engineering at Johns Hopkins University School of Medicine. Within five weeks of cultivation, the TMJ grafts contained fully viable cells at a physiologic density, forming confluent layers of lamellar bone, mineralized matrix, and osteoids.

“We take knees from cows and drill into them and get a piece of the trabecular bone on the order of millimeters in size. The bone is then decellularized in a way that minimizes the damage to the native extracellular matrix (ECM). The idea was that we could provide native signals (such as stiffness and bioactive factors) that cells would see in vivo at their normal anatomical location. In this case we were injecting mesenchymal stem cells. In a bone-like environment, the cells would be driven to differentiate into bone-like cells.”

Columbia and Johns Hopkins scientists designed a bioreactor with an anatomical culture chamber to aid in development of TMJ bone grafts.

Lung Tissue

Joan E. Nichols, Ph.D., associate professor in the departments of internal medicine and microbiology and immunology at the University of Texas Medical Branch (UTMB), discussed her lab’s work on the use of whole acellular (AC) lung as a matrix to support development of engineered lung tissue from murine embryonic stem cells (mESCs). 

Dr. Nichols explained that the design of biomaterials that can guide stem cell behavior and facilitate lung lineage choice, as well as allow seamless integration of the engineered lung tissue into living lung tissue, will require both the development of decellularized matrices, as well as an understanding of the impact of the unique lung ECM on cell behavior and function. 

Thus far, she said, attempts to develop lung tissue equivalents have used relatively simple matrices not designed to meet requirements for lung in terms of matrix composition, elasticity, or porosity. Dr. Nichols and her research partner, Joaquin Cortiella, M.D., professor in the department of anesthesiology at UTMB, have used ACM populated with mESCs to build a potentially more robust lung tissue equivalent.

Dr. Nichols explained that the ECM of any tissue supports the architecture and structure of the tissue and plays a role in development, growth, physiology, and response to injury. For matrix selection for lung tissue development, she noted that the biocompatibility, elasticity, and the adsorption kinetics of the material used are particularly important.

“Biomaterials designed for use as a matrix for regenerative medicine purposes fail to replicate the complexity of the ECM that is found in the natural lung. So the best overall choice for a matrix to support growth of lung tissue may be the natural decellularized lung itself. In our studies, we found that AC lung promoted better ESC survival, attachment to the matrix, and lung-specific differentiation.”

AC natural lung allowed, she added, for better retention of cells with more differentiation of mESCs into epithelial and endothelial lineages than was seen for synthetic matrices evaluated. “In constructs formed from mESCs and whole AC lung we saw indications of differentiating ESC organized into 3-D structures reminiscent of complex lung tissue.”

In the initial part of their study, the scientists compared growth and differentiation of mESCs to growth on AC rat lung,  Matrigel, Gelfoam, and a type I collagen matrix. In the AC-cultured mESCs, the investigators saw that significantly more of the cells were viable and had differentiated into cells found in the lung.

The investigators also reported evidence of site-specific differentiation in the trachea and distal lung. “For example, type II pneumocytes found in the distal lung formed hollow alveolar–like cysts lined by a monolayer of epithelial cells, which produced both pro-SPC, the nonsecreted form of surfactant protein C, as well as production of surfactant protein A.”

With respect to immunogenicity, Dr. Nichols said that preliminary in vitro data suggests that the matrix is nonimmunogenic and that little activation of immune cells cultured in contact with AC lung/trachea matrix derived from normal tissues occurs. “Little to no response in these types of studies suggests that rejection may not be a problem.”

“We are just beginning to understand how we can use this novel matrix material. I think that clinical applications of the matrix itself could be developed for clinical use in five to seven years. AC trachea has already been used in a clinical application in Italy, which is a good first step.”

Vascular Grafts

Todd McAllister, Ph.D., president and CEO of Cytograft Tissue Engineering, discussed the company’s technology for building vascular grafts using autologous cells. He said that Cytograft’s technology, called tissue engineering by self assembly (TESA), produces versatile tissues with high mechanical strength that are free of synthetic scaffolding or exogenous biomaterials, thereby avoiding the risk of immunologic responses or rejection.

In 2007, the company reported clinical results using its Lifeline™ graft in kidney dialysis patients in whom conventional hemodialysis access shunts had failed. Conventionally, such arteriovenous (AV) shunts are made by creating a “short circuit” between an artery and a vein to accelerate blood flow in order to shorten hemodialysis  times. These conduits, Dr. McAllister said, must withstand extraordinarily high flow rates and are punctured six times weekly during the hemodialysis sessions.

Ideally, this short circuit can be created using the patient’s own tissue by connecting a vein directly to an artery. Over time, however, the shunts fail, necessitating the use of synthetic materials such as Gortex or chemically modified animal veins to re-create the shunt. The synthetic grafts demonstrate significantly higher failure rates than native tissue.

“Despite the relatively low number of hemodialysis patients in the U.S., maintenance of hemodialysis access grafts absorbs more than 1 percent of Medicare’s entire budget, making AV access a multibillion dollar problem.”

As reported in The Lancet, Dr. McAllister and his colleagues implanted Lifeline grafts in 10 patients with end-stage renal disease who had been receiving hemodialysis through an access graft that had a high probability of failure, and who had experienced at least one previous access failure. Completely autologous tissue-engineered vascular grafts were grown in culture supplemented with bovine serum, implanted as arteriovenous shunts, and assessed for both mechanical stability during the safety phase and effectiveness after hemodialysis.

The grafts are created using fibroblasts removed from individual patient’s skin, which are then grown in tissue culture to produce a sheet composed of the cells and proteins such as collagen and elastin produced by the cells. The sheet is then rolled up into a multilaminate roll and allowed to fuse into a uniform tissue. The inner layers are then air dried and a second living sheet is wrapped around the outside. Decellularization of the inner layers prevents migration of any cells into the interior lumen of the vessel. The patient’s own endothelial cells are then added to the inside of the nascent vessel to prevent the vessel from clotting.

In the clinical trial, three grafts failed within the safety phase, consistent the company said, with expected failure rates in this high-risk population. One patient was withdrawn from the study due to unrelated gastrointestinal bleeding that occurred immediately prior to implantation and another patient died of unrelated causes during the study safety period. The five remaining patients had grafts that continued to function for hemodialysis 6 to 20 months following implantation and a total of 68 patient-months of patency. Of note, Dr. McAllister said, is that the complication rate for these surviving grafts was significantly lower compared to the standard of care.

Overall, primary patency was maintained in seven of the remaining nine patients, one month after implantation and in five of the remaining eight patients, six months after implantation.

Cytograft Tissue Engineering’s team works to produce tissue engineered blood vessels for clinical use in a GMP facility in Novato, California.

Patricia F. Dimond, Ph.D. ([email protected]), is a life science consultant.

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